Project Summary The atrioventricular junction (AVJ) plays vital roles for cardiac development and function. The AVJ myocytes provide a morphogenetic and electrophysiological boundary between the atrium and ventricle, instruct endocardial cushion formation, and recruit extra cardiac coronary stem cells to the heart. Defects in AVJ conduction delay, cushion formation, and/or extra cardiac cell entry to the heart, can all be prevalent and life threatening components of several congenital heart syndromes in humans. However, despite more than a century of investigation, the mechanisms that induce and specify the AVJ fate and function remain poorly understood. The current dogma assumes that the AVJ myocytes arise from the primary heart field and uniquely maintain molecular and physiological features of primitive heart tube myocardium. It is also postulated that a feedback loop encompassing Bmp2 and the transcription factors Tbx2/3 maintain this juvenile myocardial state, thus specifies AVJ fate. Contrary to these models, our preliminary studies show that: (i) AVJ myocytes are not derived from either the primary or secondary heart field; (ii) Slow conducting AVJ myocytes display specific electrophysiological and molecular characteristics distinct from primitive heart tube myocytes; (iii) Direct exposure of the AVJ to endoderm-derived fast conducting fate inducers up-regulate Cx40 in the AVJ myocytes and eliminate AVJ delay without suppressing either Bmp2 or Tbx2/3 expression; (iv) The AVJ can be converted to fast conducting by removal of the cardiac jelly that separates the AVJ myocytes from the endocardium; and finally (iv) AVJ myocytes pattern a pulsatile Ca2+ wave through the proepicardium from which extra cardiac coronary stem cells arise. Based on these surprising findings, this application proposes to experimentally test the following novel models for AVJ development and function: (i) Identity of AVJ myocytes is established within the newly identified Nkx2.5/ Isl1-negative tertiary heart rather than the Nkx2.5/Ist1-positive primary and secondary heart fields; (ii) The Bmp2-Tbx2/3 signaling axis acts as a morphogenetic cue, rather than a myocyte-autonomous cell fate determinant for slow conducting zone formation at the AVJ; and (iii) AVJ myocytes spatially pattern a novel pulsatile intercellular communication between proepicardial cells via a Ca2+ wave during oriented extension towards the heart. Using in vivo and in vitro approaches, this proposal will provide the first insight into the origin of the AVJ and the previously unrecognized roles of AVJ myocardiumn for proper heart development and function.